Methods for screening substances in a microwell array

ABSTRACT

Methods for manufacturing and using an apparatus for manipulating and analyzing a large number of microscopic samples of a liquid or materials, including cells, in liquid suspension. Parallel through-holes are formed in a platen and loaded with a liquid. Loading may be performed in such a way as to create a gradient, with respect to the position of the through-holes, of the concentration of a particular substance or of another quantity. Mixing of the contents of through-holes may be obtained by bringing filled microwell arrays into contact with each other with registration of individual through-holes.

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/225,583, filed Jan. 5, 1999, issued May 14, 2002as U.S. Pat. No. 6,387,331, which in turn claims priority from U.S.Provisional Application No. 60/071,179, filed Jan. 12, 1998, both ofwhich applications are herein incorporated by reference. The presentapplication claims priority, additionally, from U.S. ProvisionalApplication No. 60/239,538, filed Oct. 10, 2000, which is alsoincorporated herein by reference.

FIELD OF THE INVENTION

The present invention pertains to methods for manufacturing and usingapparatus for manipulating, transporting, and analyzing a large numberof microscopic samples of a liquid or of materials including cellscurrently or formerly in liquid suspension.

BACKGROUND OF THE INVENTION

Chemistry on the micro-scale, involving the reaction and subsequentanalysis of quantities of reagents or analytes of order microliters orsmaller, is an increasingly important aspect of the development of newsubstances in the pharmaceutical and other industries. Such reaction andanalysis may accommodate vast libraries containing as many as a millioncompounds to be reacted and analyzed under various conditions.Significant problems associated with current technologies as applied tochemical analysis of vast numbers (potentially on the order of hundredsof thousands or millions per day) of compounds include the problem ofhandling vast numbers of compounds and reactions in parallel.

Existing technology relies on 96-, 384-, or 1536-well plates containingquantities between approximately 1 microliter and 1 milliliter of liquidcompound per well, and, generally, involves chemical reactions andanalysis in wells disposed with single openings on flat surfaces such aspolystyrene. It is not practical to apply existing technology in the artto form million-well microtiter plates. There is a need, therefore, fornew approaches that permit the analysis of a million samples in alaboratory format.

SUMMARY OF THE INVENTION

In accordance with preferred embodiments of the present invention, amethod is provided for loading a plurality of disparate samplecontainers, the sample containers comprising an integral structure, suchthat the concentration of a specified substance in each container ischaracterized by a gradient with respect to position of the containerwithin the structure. The method has the steps of introducing a firstliquid into the disparate containers and contacting the containers witha second liquid, the second liquid containing the specified substance,in such a manner that the degree of diffusion of the specified substanceinto the disparate containers differs in a controlled manner among thecontainers.

In accordance with alternate embodiments of the invention, the durationof contact with the second liquid may be differed as among the disparatecontainers. The diffusion rate of the specified substance into thecontainers may be modulated. The method may have the further step ofcreating a gradient of the specified substance in the second liquid, andthe gradient may be created by applying electrophoresis. The diffusionrate may be modulated by contacting the containers with the secondliquid through a membrane with different permeability at differentportions of the membrane.

The step of introducing the liquid into containers may include loadingthe liquid into a plurality of substantially parallel through-holes in aplaten. Loading may be performed by forming a droplet of the liquid onthe end of a liquid transfer device, moving the fluid transfer device soas to drag the droplet across the top surface of the platen and over thethrough-holes to be filled, dispensing the liquid to keep the dropletfrom being depleted, and withdrawing the droplet from the surface afterthe through-holes are filled.

In accordance with further embodiments of the invention, a method isprovided for loading a liquid sample into a plurality of through-holesof a platen. The method has the steps of filling an array of capillarytubes having dispensing ends, disposing each dispensing end in proximityto a distinct through-hole, and transferring liquids to thethrough-holes of the platen through the capillary tubes. An alternatemethod has steps of creating regions of distinct concentration of thespecified substance in a liquid, such as by electrophoresis, andtransferring into distinct containers the liquid from the distinctregions. The liquid may be transferred through capillary tubes or bycontacting the structure containing the through-holes with the surfaceof the liquid.

In accordance with other embodiments of the invention, a method isprovided for manufacturing a platen having two substantially parallelplanar surfaces and a plurality of through-holes disposed substantiallyperpendicularly to the planar surfaces. The method has the steps ofproviding a sheet of thermoplastic material, loading the sheet ofthermoplastic material into contact with a surface of a die having aplurality of holes, and bringing a punch having a plurality ofprotrusions of specified cross-section into contact with the sheet ofthermoplastic material in such a manner that the protrusions are inalignment with the holes of the die such that through-holes are cutthrough the thermoplastic material. In an alternate embodiment of theinvention, a sheet of electrically conducting material is loaded intocontact with a surface of a die having a plurality of holes and an EDMmandrel having a plurality of protrusions of specified cross-section isbrought into proximity of the sheet of conducting material in such amanner that the protrusions are in alignment with the holes of the die,and through-holes are cut through the conducting material.

Another embodiment of the invention provides a method for applying ahydrophobic coating to a silicon platen having a first and a secondsurface, the surfaces being substantially parallel, and a plurality ofthrough-holes substantially perpendicular to the surfaces. The methodhas the steps of oxidizing the first surface, cleaning the oxidizedfirst surface, applying a positive pressure of inert gas to theplurality of through-holes from the direction of the second surface, andexposing the first surface to a silanizing vapor agent.

In accordance with other alternate embodiments of the invention, amethod is provided for loading a liquid into a plurality of through-holeplatens having the steps of stacking at least two platens together insuch an adjacent manner that at least one of the plurality ofthrough-holes from each platen is registered with a through-hole of eachother adjacent platen so as to form at least one continuous channel, andtransfering the liquid into each continuous channel. Each platen may beseparated from each adjacent platen by an air gap, and the liquid may betransferred with capillary tubes or at least one cannula.

A method for mixing liquid that is contained in through-holes of atleast two platens is provided that includes stacking the platenstogether for a specified time, in such a manner that at least one of theplurality of through-holes from each platen connects with acorresponding through-hole of another platen and liquid is allowed todiffuse between connecting through-holes. The platens may then beseparated after the mixing.

In accordance with yet further alternate embodiments of the invention, amethod for humidifying a system is provided, where the method has thesteps of filling a microchannel plate with a liquid having a pluralityof parallel microchannels and placing the filled microchannel plate invicinity of the system to be humidified.

Another alternate embodiment of the invention provides a method fordiffusing light. According to this method, a fluid is entrained insubstantially each of a plurality of parallel microchannels havingproximal and distal ends, the microchannels comprising a microchannelplate. The proximal end of each of the plurality of parallelmicrochannels is illuminated with light and diffuse light emanates fromthe distal ends of the microchannel.

In accordance with yet another embodiment of the invention, a perforatedplaten is provided for manipulating distinct liquid samples of volumeless than 1 microliter. The platen has an inner layer of hydrophilicmaterial and two outer layers of hydrophobic material coupled toopposite sides of the inner layer, and a two-dimensional array ofthrough-holes, at least two holes having distinct volumes, for retainingthe distinct liquid samples, the through-holes each having a diameterless than 300 micrometers and traversing the inner layer and the twoouter layers in a direction substantially perpendicular to each of twoplanar surfaces of the platen.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more readily understood by reference to thefollowing description, taken with the accompanying drawings, in which:

FIGS. 1 a and 1 b show a top and exploded cross-sectional views,respectively, of a high-density array of through-holes in accordancewith one embodiment of the present invention;

FIG. 1 c shows a schematic side view of a continuous sheet array ofthrough-holes in accordance with another embodiment of the presentinvention;

FIG. 2 a is top view of a portion of the platen of FIG. 1 a in which thethrough-holes are configured on rectangular centers;

FIG. 2 b is top view of a portion of the platen of FIG. 1 a in which thethrough-holes are configured in a hexagonal close-packed array;

FIG. 3 is a further side view in cross-section of a portion of alaminated platen containing multiple through-holes for analysis ofliquid samples in accordance with a preferred embodiment of the presentinvention;

FIG. 4 is a top view of round sample wafer populated with through-holesin accordance with an embodiment of the present invention;

FIGS. 5 a-5 d show examples of arrays with through-hole volumes that area function of array position;

FIGS. 5 e and 5 f show cross sections of through-hole prisms as employedfor massively parallel liquid chromatography or electrophoresis, inaccordance with embodiments of the present invention;

FIG. 6 shows an example of an interlocking array;

FIG. 7 depicts the configuration of a compression mold for compressionmolding on an array of through-holes in accordance with an embodiment ofthe present invention;

FIG. 8 is a flow chart depicting steps in the fabrication of athrough-hole array by EDM, in accordance with an embodiment of theinvention;

FIGS. 9 a-9 c depict a sequence of operations for filling a through-holearray with a first liquid;

FIG. 10 is a schematic depiction of a method for sampling the contentsof a large-format microtiter plate and loading a subarray of thethrough-hole array in accordance with an embodiment of the presentinvention;

FIG. 11 is a schematic depiction of a method for sampling the contentsof a large-format microtiter plate and loading subarrays of multiplethrough-hole arrays in accordance with an embodiment of the presentinvention;

FIG. 12 a depicts parallel loading of subarrays of multiple stackedmicrowell arrays in accordance with embodiments of the presentinvention;

FIGS. 12 b and 12 c depict parallel loading of subarrays of a microwellarray by means of flexible members, in accordance with embodiments ofthe present invention;

FIG. 13 a is a flow chart depicting a method for successive dilution ofthe contents of a microwell array in accordance with an embodiment ofthe present invention;

FIGS. 14 a-14 c depict a sequence of operations for exposing respectivethrough-holes of a through-hole array to a second liquid and forcreating a specified gradient of a specified characteristic with respectto placement of the through-holes in the array in accordance with anembodiment of the present invention;

FIG. 14 d shows a side view is shown of a through-hole array plate witha graduated filter for providing a concentration gradient in accordancewith an embodiment of the invention;

FIG. 15 depicts mixing of the contents of a through-hole array with thecontents of another through-hole array brought into registration of thethrough-holes in accordance with embodiments of the present invention;

FIGS. 16 and 17 depict further stages in the mixing of the contents of athrough-hole array with the contents of another through-hole arraybrought into registration of the through-holes in accordance withembodiments of the present invention;

FIG. 18 depicts mixing of the contents of a through-hole array with thecontents of another through-hole array by application of externalpressure, in accordance with embodiments of the present invention;

FIG. 19 depicts mixing of the contents of a fully filled through-holearray with the contents of another incompletely filled through-holearray brought into registration of the through-holes in accordance withembodiments of the present invention;

FIG. 20 shows plots of calculated times for evaporation of a waterdroplet as a function of the ambient relative humidity;

FIG. 21 depicts a humidity chamber for loading and unloadingthrough-holes while maintaining a high relative humidity in theenvironment surrounding the array, in accordance with an embodiment ofthe present invention;

FIG. 22 a depicts an exploded perspective view of a portable humiditychamber for preventing evaporation of fluid from the arrays duringfluorescent imaging analysis, incubation and transferring the arraybetween other humidified environments; and

FIG. 22 b is a cross-sectional side view of the a portable humiditychamber of FIG. 22 a.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The Through-Hole Array

In accordance with the present invention, methods are provided forproducing different chemical reactions within an array of through-holes.The invention is advantageously employed, for example, in screeningoperations, where different reaction conditions are advantageouslyprovided among the various through-holes of the array. As an example ofthe many modalities of use of the invention, different chemical speciesmay be loaded into different through-holes of the array, andconcentrations of the various species might also be differentiated amongthe various through-holes. The invention may thus provide a method forscreening compound libraries, for example, to predict the ability ofeach compound to be absorbed by a patient.

In accordance with preferred embodiments of the invention, ahigh-density array of through-holes is provided, as now discussed withreference to FIGS. 1 a and 1 b. FIG. 1 a shows a top view of a platen10, otherwise referred to herein as a “substrate,” “sample wafer,” or“through-hole plate.” Platen 10 is the carrier of a large number ofthrough-holes 12 which traverse platen 10 from a top surface 14 to anopposing surface 16 of the platen, as shown in the cross-sectional sideview of FIG. 1 b. While the term “platen” may refer to a structurehaving substantially parallel plane surfaces and transverse dimensionssubstantially exceeding the thickness of the structure, alternativegeometries are within the scope of the present invention, and use of theterm “platen” is not restrictive. A prism-shaped geometry is describedbelow, for example.

Through-holes 12 constitute assay wells (or “microwells”) in accordancewith an embodiment of the invention. Through-holes 12 may be shaped ascircular right cylinders, or, alternatively, may have rectangularcross-sections. Otherwise-shaped through-holes are also within the scopeof the present invention.

While through-hole plate 10 is preferably made of conductive silicon,other types of rigid materials, such as metal, glass, or plastic may beused provided that the material is chemically inert with respect to thesample substances, or can be rendered so by appropriate surfacetreatments.

Each through-hole 12 is typically of a substantially squarecross-section, although geometries, such as circular or rectangularcross-sections may be used. Through-holes 12 are also referred to hereinas “channels.”

Through-holes 12 may be centered on a rectangular grid, as shown in FIG.2 a, or in a close-packed hexagonal lattice, as shown in FIG. 2 b.Referring to FIG. 3, a typical thickness 20 of platen 10 is on the orderof 0.5-2 mm, while through-holes 12 have typical characteristicdimensions (such as diameters) 36 of on the order of 100-400 μm. Thusthe volume of each through-hole 12 between surface 14 and surface 16 ison the order of ˜10⁻⁷ cm³ or greater. Through-holes 12 are spaced oncenters typically on the order of twice the diameter of the holes,although all spacing configurations are within the scope of theinvention and of the appended claims. In particular, a hole-to-holespacing 38 of 500 μm is typically employed, which corresponds to anarray density of 400 holes per square centimeter of plate. In accordancewith manufacturing methods described below, microwells are produced forthe assay of a chemical or biochemical reaction where the volume of eachmicrowell may be less than 100 nanoliters (10⁻⁷ cm³). The packingdensity of wells may thereby be increased by several orders of magnitudeover prior art technology.

Grouping of through-holes into smaller sub-arrays may also be used, and,more particularly, a reproducible pattern may be applied to a pluralityof the sub-arrays. Each through-hole 12 may be identified by its ownaddress within the array.

Referring, again, to FIG. 1 b, platen 10 may also advantageously beformed of a laminate of materials, with a central layer 24 and outer“sandwiching” layers 22. In order to enhance capillary loading of sample30 into the microwell and to prevent capillary outmigration of thesample liquid, exterior sections 22 of the microwell, adjacent tosurfaces 14 and 16 of platen 10, have a hydrophobic wall surface inaccordance with a preferred embodiment of the invention, while theinterior section 24 of the through-hole wall has a hydrophilic surfacethereby preferentially attracting an aqueous liquid sample. In a similarmanner, an array having hydrophilic faces and hydrophobic through-holesmay be uniformly filled with a low surface tension liquid such as analkane. The hydrophobic layers on either end of the well are on theorder of 1 μm thick or less. On loading the sample liquid into themicrowells, each well is typically overfilled by about 10% above thevolume surrounded by the four walls of the microwell. Under thesecircumstances, liquid sample 30 may form convex meniscus surfaces 34 onboth the upper and lower surfaces of the sample.

An underfilled microwell 26 will typically be characterized by a liquidsample exhibiting a concave meniscus 28 on both the upper and lowersurfaces of liquid sample 30.

The apertures of through-holes 12 need not be square, and, in accordancewith an alternate embodiment of the present invention, flanges 8 mayextend above planar surface 14 surrounding some or all of through-holes12 while indentations 6 may be fabricated rounding the edges ofthrough-holes 12 at opposing surface 16. Flanges 8 and indentations 6may advantageously provide for registration of successive platens 10, inthe case where platens are stacked, and in processes of mixing ordilution, as discussed in detail below.

Through-holes 12 may be loaded with a first sample 18 in liquid form.Sample 18 is allowed to react with a second sample where the secondsample may include a variety of test samples and by subsequent orconcurrent analysis of the reaction products, using, for example,optical markers, a large number of reactions may be processed andanalyzed in parallel.

As applied to biological assays, by way of example, first sample 18 maybe a solution containing pharmacologically relevant proteins or othermolecules. Such a solution may include, for example, cells in aqueoussuspension, eukaryotic (animal, yeast) or prokaryotic (bacteria) cells,hybrid cells, and biological molecules including, for example,antibodies and enzymes, although application to other biological ornon-biological assays is within the scope of the invention as claimedherein. All such reagents may also be referred to herein and in theappended claims as “targets.” Typical yeast cell concentrations of 10⁷cells per milliliter of solution yield on the order of 1000 cells per100 nanoliter well. Typically, an entire chip or the subset ofthrough-hole wells constituting a contiguous region of platen 10 may bepopulated with a single strain of cells.

A typical procedure assay procedure, such as may be employed inpharmaceutical research, entails the subsequent addressed introductionof a test sample including one or more analytes into the through-holewells, with selected materials introduced into subsets of through-holesthat may include one or more through-holes. The test sample addressablyintroduced into the subsets of through-holes may contain drug candidatesor known drugs. The test sample may be comprised of multiple components,introduced at the same time or sequentially. Components of the testsample may include analytes, antagonists, reagents, solvents, or anyother materials and may be introduced in liquid form or otherwise. Inaccordance with a preferred embodiment of the invention, test samplesare introduced into the through-hole wells in liquid form in order tofacilitate rapid reaction via diffusion with first sample 18 alreadyresident in liquid form in the through-holes.

The set of substances from which the second sample addressed to aparticular through-hole site is drawn is referred to in this descriptionand in the appended claims as a “library” of substances. In typicalapplications, the library is of a substantial size and thusadvantageously utilizes the capability of the present invention tofacilitate parallel reaction and analysis of large numbers ofsubstances. In pharmaceutical applications in particular, libraries maybe composed of between 10³ and 10⁹ substances and combinations ofsubstances.

Referring now to FIG. 1 c, the throughput of a screening systemincorporating arrays of through-hole sample containers may be furtherincreased by using a continuous sheet 300 of through-holes 12. Theinteriors of the through-holes may be hydrophilic, as described above inreference to FIG. 1 b, and the surface of sheet 300 may be hydrophobic,as also described. Through-holes 12 may be filled in a continuous mannerby passing sheet 300 through an aqueous medium 302 contained in fluidtrough 304. After through-holes 12 have been filled, sheet 300 may bewound onto a spool 306 or cassette for storage followed by assay, or,alternatively, may be assayed directly. Assays may be by optical readoutsuch as fluorescence absorbance or chemi-luminescence measurements, allof which may be performed by passing the sheet across an opticaldetector such as a CCD array.

Sample array sheets 300 are preferably produced with registered holes,either by precision production processes or in matched sets. The sheetcomposition may be a polymer, elastomer, or metal, including anamorphous metal. Multiple sheets may be mated in the same way thatplatens of through-holes may be stacked, as described below, forexample, with reference to FIGS. 16-19, in order to initiate reactions,or for other purposes. FIG. 1 c shows a second sheet 308 ofthrough-holes 310 being brought into contact with sheet 300 in mixingarea 312 for mixing of the liquid contents of the respectivethrough-holes. Examples of applications involving this embodimentinclude, screening genetic libraries or screening combinatorial chemicallibraries contained on polymer beads. These embodiments of the inventionmay advantageously include extremely high throughput, reduction orelimination of high-cost automation components, and the small size of ascreening system with sample handling and detection modules. The holesin the sheet may be, if desired, produced online with an array ofpunches or UV lasers.

As an example of an application in which a genetic library is screenedfor improved enzymes using a one step assay, an E. coli genetic libraryis prepared containing mutations in the beta galactosidase enzyme. TheE. coli cells are grown to a density in phosphate-limited media suchthat there is an average of 1 cell for every 200 nl of liquid. The mediaalso contains, MUG, a fluorogenic substrate for beta-galactosidase. Athrough-hole sheet is prepared with a hydrophobic exterior andhydrophilic through-holes at a density of 10^7 per square meter.Registration holes are includes in the tape to aid in precisedispensing. Each through-hole holds 70 nl of fluid. A spool of thethrough-hole sheet is unwound and guided through a trough containing thecell solution, so that each through hole is filled. The sheet is thenwound onto spacers and into a receiving spool. The spacers preventsmearing of the liquid and provide for gas transfer in and out of thespool. The spool is incubated in a humidified enviroment at 37° C. for24 hours. The spool is then unwound as it is passed between a uniformphoto-illumination source with a wavelength of 350 nm and a CCD imagingsystem with a 450 nm filter. The position in the sheet of colonies withexceptionally high enzyme activity is recorded and those colonies areretrieved using a robotic microfluid handling system for furtheranalysis. This assay can also be performed by co-registering and matinga second, identical, sheet containing the fluorogenic substrate with thefirst sheet containing the bacteria. An absorbance measurement may alsobe performed to normalize the signal output for the number of bacteria.

In accordance with another alternate embodiment of the presentinvention, described with reference to FIG. 3, through-holes 12 may bedisposed in an array within a circular sample wafer 320 having a centralhole 322 for purposes of centering with respect to handling equipment.

Referring now to FIG. 5, platen 10 and through-holes 12 may assume othergeometries than have heretofore been described, in accordance withalternate embodiments of the invention. The volumes of the through-holesmay be varied as a known function of spatial location in the array. Thevolume of a through-hole V=k s²l, is a function of lateral holedimension, s, length, l and constant k that depends on the specificcross-sectional hole geometry. The cross section may be varied, asdepicted in FIG. 5 a, as a known function of spatial location within thearray by changing the hole's lateral dimensions as a function ofposition within the array. The hole volume scales as s²; thus increasingthe hole dimensions by a factor of 3.3 increases the volume by a factorof 10. One example is in a plane parallel plate, the hole dimensions areincreased as a linear function in one or both lateral directions.

Another method to produce different through-hole volumes as a functionof array position is to change the distance between top and bottomsurfaces of the plate defining the hole length 52, as shown in FIG. 5 b.One particular example is to incline the plate top 14 and bottom 16surfaces at an angle to each other as in a wedge or prism, as shown inFIG. 5 c. In this example, the volume change is linear with distancealong the array in the direction of surface inclination while in theorthogonal direction along a row of holes, the hole length, and thusvolume, is constant. In accordance with another alternate embodiment ofthe invention, an array can be fabricated from a contiguous series ofplanar surfaces inclined to each other in either one- or two-dimensionssuch that the through-hole volume is different in both directions alongthe array. In yet a further embodiment, an array is fabricated from anon-planar surface 54 such as a surface of hemispherical curvature asshown in FIG. 5 d.

For each array geometry, a second array fabricated having surfaces thatare the complement of the first array's geometry may be used, inaccordance with alternate embodiments of the invention, to facilitatesvertical stacking of the arrays, as shown in FIG. 6. Alignment ofthrough-holes 64 of plate 60 with through-holes 66 of plate 62 isprovided by inserting protruding segments 68 into correspondingindentations 70. Other array geometries with greater than four-foldrotation symmetry with respect to the array surface normal willfacilitate interlocked and self-aligned stacking of arrays with matchingpositive and negative geometries, within the scope of the presentinvention. This geometrical arrangement may advantageously obviate theneed for alignment pins which are typically required for registration ofstacked planar arrays.

Applications of Arrays of Non-Uniform Volume

One application of arrays having through-holes of non-uniform volume ismixing of different volumes of liquids such as in a dilution sequence,as described below. An advantage of this method is that stacking ofmultiple arrays, as also described below, may be obviated while a widerange of dilution may be achieved.

Another application of arrays of either constant or non-uniformthrough-hole volumes is fraction collection from chromatographicelution. The through-hole array advantageously provides the ability tocollect a large number of small volume fractions, which can then befurther separated within the through-hole array, as described below.This, in turn, advantageously increases the resolution ofchromatographic separation over prior technology.

Yet another application of non-standard array geometries is formassively parallel liquid chromatography or electrophoresis. Moreparticularly, referring to FIG. 5 e, a through-hole plate 56 is employedwhose thickness increases in one lateral direction (such that the platehas the shape of a prism) resulting in a linear increase in hole lengthwith array position. A second through-hole prism 57 is brought togetherwith the first prism 56 along their common hypotenuse 58 therebyproducing an array of through-holes 12 of substantially equal length. Touse this structure as a liquid chromatography column, each hole isfilled with a porous gel 55 characteristic of liquid chromatography. Thesample 51 to be analyzed is applied to one end of the array, as from asample plate 53, for example, and a pressure is applied to drive fluidsample 51 through each hole. Each component in the mixture will travelat a different velocity through the gel matrix resulting in a separationof the mixture along the column length. For example, small moleculeswill move rapidly through the channel to its opposite end, whereas,after the same duration of time, larger molecules may typically havetraveled only a fraction of that distance. The intersection of eachthrough-hole with the hypotenuse along which the prisms are joinedcorresponds to different travel lengths along the chromatographiccolumn. Separation of the array into its constituent prisms, as depictedin FIG. 5 f, gives a “snapshot” of the mixture components distributed asa function of array position. Rather than a function of time, thechromatograph is transformed into a function of position by runningidentical samples in an array of columns with a known inter-columndelay, expressed either as a time, or, equivalently, as a length. Bysequentially analyzing the material output from each through-hole alongthe direction of increasing plate thickness, an equivalent chromatographcan be reconstructed. Further mixture separation is possible if the gelporosity is made different in the array direction orthogonal to thewedge orientation. Decreasing gel porosity further increases theretention time for each component, thus leading to finer resolution ofthe mixture components. Electrophoretic separation may be achieved in ananalogous manner, where an applied electric field is the driving force.

Fabrication of the Through-Hole Array

Referring now to FIG. 7, through-holes 12 may be formed in platen 10 byany of a variety of means appropriate to the material of platen 10.Through-hole forming methods include, by way of example, laser ablationby means of an ultraviolet (UV) excimer laser which may form 100 μmthrough-holes in glasses and polymers. Additional through-hole formingtechniques include mechanical drilling, electrochemical methods such asmicro-electrical spark discharge machining (EDM), employingradio-frequency pulses for ionizing removal of conductive materials, or,in accordance with other embodiments of the invention, by selectivechemical or charged-particle etching techniques. Additionally,microcapillary bundles of glass fibers of varying compositions may bedrawn from preform and sliced to form platens, and then selectivelyetched to form through-holes.

As shown in FIG. 7, through-holes 12 may be formed, in materials such asthermoplastics or polycarbide, by punching platen blank 400 using puncharray 402 and die array 404, in conjunction with a high-pressure ram.Punching pins 406 may be formed in punch block 408 using microwire EDM,as described in detain in the following discussion, or by microetchingtechniques such as chemical or charged-particle etching. Additionally,punch array 402 may be formed using microsawing techniques. Die array404 is similarly formed using microfabrication techniques known in themanufacturing arts.

High-density arrays of through-holes may be formed in conductingmaterials, including conductive (>10 Ω⁻¹-cm⁻¹) silicon wafers, using acombination of wire and die sink EDM. EDM is typically used in thepreparation of tooling dies for injection molding. As is well known, themachining process involves ionizing away the surface of a conductingmaterial, typically a metal. EDM can be performed using a tip-basedelectrode, or a “wire.” Wire-EDM is used when fine surface finishes arerequired, or when subtle machining features not achievable with a tipelectrode are desired. Traditional wire-EDM machining utilizes wireapproximately 250 μm in diameter. Due to the small dimensions and highpacking densities of the invention an adapted microwire EMD process isused that employs wires with diameters down to 30 μm. This system mayprovide surface finishes down to 100 nm, essentially a mirror finish.

Referring to FIG. 8, a typical method for preparing a through-hole arrayusing microEDM machining follows a two-step process. The first step isthe creation of a positive or master die. The second step uses anotherEDM machine called a “sink-EDM.” The sink-EDM machine uses the master asan electrode and thereby creates a negative copy in the machinedconducting material. This negative copy is the resulting microarray. Themaster electrode can produce multiple negatives before needing to bereplaced, thereby increasing manufacturing throughput for production ofthe chips.

In contrast with deep reactive ion etching—the process commonly used toproduce high aspect ratio structures in silicon—the EDM techniquedescribed herein may advantageously reduce fabrication time and cost.

After the master die is fabricated with wire EDM, it may typically beused to sink an array of microchannels through a 0.5-mm thick siliconwafer in less than two minutes. Additional machining time, during whichthe master die moves largely in the plane of the array, is typicallyneeded to enlarge the holes to the desired dimension and to improve thesurface finish. The fabrication process described has been used to sinkmicrochannels of sizes up to 10,000 elements with cross-sections <300μm×300 μm and element spacings <500 μm. Another advantage of thistechnique is that it may also be used to a manufacture stainless steelprecision alignment jig that is used to align the chips for mixing andoptical readout, as described herein.

Combined with the precision alignment jig, this EDM process results in<10 μm center-to-center total error in channel spacing across the entirearray. This guarantees accurate hole alignment across arrays, and whencombined with the hydrophobic exterior coating, minimizes cross talkbetween microchannels.

Referring again to FIG. 7, the master die 408 produced from the processoutlined above can be used to create the through-hole arrays from aplastic blank 400. In this approach the die is used as a type of punchto create the through-hole array from a solid piece of plastic. Unlike apunch, however, the master does not force its way through the plastic.Instead, by having the appropriate kinetic energy as it impacts theplastic it essentially vaporizes the solid plastic into low molecularweight gases (with some residual energy dissipated as heat). In thismanner the master can be removed from the resulting through-holeswithout having melted into it. A slight taper on the protrusions of themaster that forms the channels facilitates removal of the master fromthe chip. This process is similar to that which is used in themanufacturing of DVD's, except the manufacture of microwell arraystypically requires significantly greater penetration depths (up to 1 mmdeep). Alternatively, plastic microwell plates may be manufactured byinjection molding of metal masters formed by EDM.

As discussed above, with reference to FIG. 1 b, it is desirable toprevent cross-communication between the various through-holes duringloading and other operations by coating the surfaces of the platen witha hydrophobic coating 22. It is also desirable to coat the innersurfaces of the through-holes with a hydrophilic coating 24 so that theyretain fluids. In accordance with embodiment s of the invention, theinner coating 24 may be chemically blocked to prevent non-specificbinding or derivatized with affinity ligands.

In accordance with preferred embodiments of the invention, a dense array10 of through-holes 12 is produced in silicon and coated in siliconoxide by oxidation. The surfaces of the conductive silicon are coveredin a thin oxide layer. The wafer is then cleaned by soaking in a mixtureof hydrogen peroxide and sulfuric acid, or other caustic solventcleaning solution, to remove organic materials. Clean silicon oxide thusproduced has a high surface energy. The top and bottom faces of thearrays are made hydrophobic by exposing them to vapor from a solutioncontaining an appropriate silanizing agent (such aspolydimethylsiloxane, sold as Glassclad 216™ by United ChemicalTechnologies, Inc.) in a volatile solvent. The silanizing agent reactswith the hydroxyl and silanol groups on the array surface and createscovalent bonds to hydrophobic alkyl groups.

The resulting coated arrays can be uniformly loaded with aqueoussolutions by simply dipping the array into it. The liquidinstantaneously fills the channels through capillary pressure, but doesnot wet the other surfaces. Hydrophobic coatings produced in this wayare stable under high humidity and they can be used repeatedly overseveral days. Other surface chemistries may be exploited to attachhydrophobic chemical groups to the faces of arrays made from othermaterials. For example a gold-coated surface reacts with alkane thiolsto attach a hydrophobic alkyl groups, as discussed by C. D. Bain et al.,J. Am. Chem. Soc., vol. 111, pp. 321-325 (1989), which reference isincorporated herein by reference.

In accordance with other embodiments of the present invention, thesurface chemistry of the array faces may also be selectively modified bysubstituting other silanizing agents for the polydimethylsiloxanetelomer. This method may advantageously prevent aqueous solutions fromadhering to array faces during array loading and also act as physicalbarriers between the aqueous solutions in adjacent through-holes. Duringthis process a positive pressure of inert gas is applied to the oppositeside of the array. The positive pressure within the through-holesprevents the silanizing vapor from reaching the interior surfaces. Thismethod advantageously allows removal and reaplication of the hydrophobiccoating.

Array Loading Techniques

Dip Loading

Referring now to FIGS. 9 a-9 c, array 10 of through-holes 12 may befilled using techniques either that directly address particularthrough-holes, or techniques that fill the entirety of the arrayaccording to a specified pattern based on composition, concentration ofa substance, etc. Dip loading may be employed, for example, in order tofill an entire array with the same solution. A through-hole array 10 isproduced and chemically treated to make the array faces hydrophobic andthe through-hole surfaces hydrophilic. Plate 10 with through-holes 12 isfirst lowered into a container 90 containing a first liquid 82, as shownin FIG. 9 a. Once plate 10 is fully immersed in the first liquid, it isshaken so that first liquid 82 displaces the air in each of thethrough-holes, and all the through-holes 12 are filled with first liquid82. All means of replacing the air in the through-holes is within thescope of the present invention, whether by shaking, applying a vacuum toone side of plate 10, employing electrostatic forces, tilting the plateso that the air is displaced by virtue of its buoyancy, all cited asexamples and without limitation, or else by other means. Afterthrough-holes 12 have been filled, plate 10 is withdrawn from firstliquid 82, as shown in FIG. 9 c. The array is typically withdrawn slowlysuch that the surface of the fluid in the reservoir pulls excess liquidoff of the non-wetting array surface. Alternatively the array may befilled with water by spraying the array with water or by sonicating thearray in a reservoir of water in order to remove trapped air bubbleprior to dip loading. In this case, less agitation is required touniformly fill the array with solution.

Both convection and diffusion can be used to induce mixing betweenfluids in microchannel arrays. This can be demonstrated by filling amicroarray with aqueous solution of blue dye and by submerging themicroarray into a beaker of still water to induce dilution viadiffusion. A small mechanical disturbance (such as a tap to the side ofthe beaker) causes rapid replacement of the blue dye by water.Alternative methods for filling the array with water include sprayingthe array with water or sonicating the array in a reservoir of water inorder to remove trapped air bubble prior to dip loading are also withinthe scope of the present invention.

Similarly, an array having hydrophilic faces and hydrophobicthrough-holes may be uniformly filled with a low surface tension liquidsuch as an alkane.

Loading by Dragging Droplet Along Array Surface

Droplet dragging is performed to load an entire array or a group ofchannels within a single array with the same fluid. It is appropriatefor loading samples which must be conserved because of cost oravailability. To perform a loading operation by dragging, a droplet ofthe loading solution is formed on the end of a syringe needle,micropipette or other fluid dispensing capillary. The drop is placed onthe array face over the through-holes to be filled. The capillary isthen moved to drag the drop across the surface of the array and over thechannels to be filled. Surface tension maintains contact between thecapillary tip and the fluid drop. When the drop is depleted additionalfluid is dispensed until all desired through-holes have been filled. Thecapillary and any remaining fluid in the drop are then withdrawn fromthe array surface.

Transfer of Liquids between Microtiter Plates to Through-hole Array

In accordance with further embodiments of the invention, a method isprovided to transfer liquids from standard microtiter plate formats(96-, 384-, or 1536-wells, for example) to a single array ofthrough-holes or multiple, vertically stacked arrays in which holes inthe same spatial location from one array to the next are co-registered.As used herein, the term “registration” refers to aligning athrough-hole plate with at least one other through-hole plate, such thatthe tops of a plurality of through-holes of one of the plates coincidewith the bottoms of a plurality of corresponding through-holes of theother plate or plates, thereby creating a plurality of contiguouschannels.

All of the filling examples given can be performed in parallel on asingle array stack with multiple capillary tubing arrays filled withliquids from different microtiter plates or the same microtiter plate.Fluid may be transferred from chemical libraries stored in 96- and384-well microfiter plates quite rapidly with these methods. Forexample, if 100 arrays with 10⁵ through-holes per array are to be filledwith liquid stored in 96 well plates, and if each transfer operation(plate exchange, fluid loading and transfer) takes approximately 20s,then the requisite arrays may be filled within approximately six hours.The filling operations take place preferably in anenvironmentally-controlled chamber (temperature, humidity, atmosphere,etc.), as described below.

Transfer of Liquids from Microtiter Plates to One or more Through-holeArray

Referring now to FIGS. 10 and 11, further embodiments of the inventionprovide an apparatus and methods that are particularly suited to thetransfer of liquids from standard microtiter plate formats (such as 96-,384-,or -1,536-well formats) to a single array of through-holes ormultiple, vertically stacked arrays in which holes in the same spatiallocation from one array to the next are co-registered. Producing suchstacks of arrays may be employed advantageously for producing replicatesof molecular or cellular libraries.

Capillary Tube Array

Viewed in cross-section, a capillary tube array 100 is constructed fromcapillary tubing 102 with an external diameter that fits precisely intothe through-holes of a through-hole microwell array 10. Tubing array 100is designed such that tubing 102 at one end 104 has a center-to-centerspacing 106 equal to the spacing between holes in through-hole array 10(or, alternatively, to an integral multiple of the inter-hole spacing)and tubing at the opposite end 108 has a center-to-center spacing equalto the center-to-center spacing 110 of wells 112 in a microtiter plate114. Plates 116, 118, and 120, with through-holes having these andintermediate separations serve as jigs to hold the tubing in a regulararray. Additional through-hole plates 116, 118, and 120 placed betweenthe two ends may advantageously serve as spacer jigs providingadditional support for the tubing array as the center-to-center spacingvaries over the tubing length.

In addition to filling a single through-hole microwell array 10, thetechnique depicted in FIG. 10 may be advantageously employed for fillingan entire stack 122 of hole-registered microwell arrays. The internalvolume of each tube 102 in the capillary array 100 is slightly greaterthan the total volume of a column of aligned holes in the array stack122. For example, if the through-hole dimensions in the array are 250μm×1000 μm, giving a volume per through-hole equal to 62.5 nl, then thevolume of one set of holes in a stack of 100 arrays is 6.25 μl (100×.62.5 nl). Capillary tubing with an internal diameter of 200 μm and anexternal diameter of 245 μm is readily available; thus a minimum tubelength of 200 mm stores the volume of fluid needed to fill this set ofthrough-holes.

One end 108 of tubing array 100 is inserted into the wells of amicrotiter plate 114, each tube being inserted into a corresponding well112. Next, a negative pressure difference is applied across the lengthof tubing 102 to draw liquid, in direction 126, from each well into itscorresponding tube. Negative pressure could be applied to each tubeindividually, or as shown in FIG. 10, the ends of the tube array canterminate in a chamber 124 that can be partially evacuated. After filingeach tube of the array, the microtiter plate 114 is removed. The liquidcan be stored in the tubing array for an indefinite period of time,either frozen or in a humidified environment. It can also be readilytransported to another location in this format. Multiple tubing arrayscan be filled from the same microtiter plate (assuming there issufficient volume of liquid per well) or different tubing arrays can befilled from different microtiter plates.

Proximity Filling

Embodiments of the invention also provide for methods for filling astack of arrays, as shown in cross-section in FIG. 11, by bringing theend of a tubing array 130 into close proximity with a matching set ofthrough-holes in the array stack 122. The tubing array can be alignedrelative to the array stack by an alignment plate 128 with through-holeshaving the same center-to-center spacing as the through-holes into whichfluid is placed. Each array 10 in the stack 122 is spaced a smalldistance s that may be, but is not limited to, an equal distance to thethrough-hole spacing. Application of pressure to the end of the tubingarray, placed inside a pressurized container 132, forces fluid from eachcapillary tube 102 into the opposing through-hole. After thethrough-hole is filled, a liquid drop can begin to grow in the spacebetween the two plates. When the drop reaches a size that it contactsthe through-hole in the plate above it, surface tension draws some fluidinto the through-hole. Once the fluidic bridge is established, liquidcan flow into the through-hole, driven by the constant pressure appliedto the opposite end of the tubing array. With no applied pressure, thedrop retreats into the through-hole, the fluidic bridge between eachplate is broken, and the separation of array plates after filling can befacilitated (i.e., because there is generally no surface tension thatneeds to be overcome). Successive filled plates 10 are then withdrawn,and the tube array may be retracted in direction 128. Each verticallyregistered set of through-holes may thus act as a channel for fluidflow. The hydrophobic coatings on the exterior surface of the arraysprevent liquid from flowing into adjacent holes. This technique can alsoadvantageously be used to create replica plates of a cell library byapplying a cell suspension with a pressure uniformly to the array stack.

Inter-hole Spacing Matched to a Microtiter Plate

A method for filling a through-hole array stack, in accordance withalternate embodiments of the invention, is shown in FIG. 12. This methoduses a through-hole array 10 having the same lateral dimensions as amicrotiter plate 114 and having a hole spacing that is an integralfraction of the well spacing in the microtiter plate 114. When the arrayis placed on top of the plate, one or more through-holes 12 align withrespect to each well 112 in microtiter plate 114. An array 130 ofsyringes 132 with a center-to-center spacing equal to the well spacingcan thus be positioned over a stack of through-holes registered withrespect to each other and the microtiter plate. The syringe array isinserted through a through-hole plate 134, such that the plate is amechanical guide for the syringe tubing as it is moved relative to thearray stack. The syringe plungers are mechanically coupled and actuatedby a mechanical or electromechanical driver module 136, such that liquidis drawn into or expelled from each syringe in parallel. The syringetubing outside diameter is preferably sized relative to the through-holelateral dimensions to give a sliding fit, and the tubing can have alength suitable to allow insertion through the array stack 122 and intothe liquid contained in the microtiter plate wells 112.

The volume of liquid withdrawn into each syringe preferably equals thevolume of liquid in a column of aligned through-holes in the arraystack. The liquid can then be dispensed as the syringe array isretracted from the array stack, and the rate of dispensing can besynchronized with the rate of withdrawal, such that each through-holeaddressed by the syringe array is filled. Once this operation iscompleted, either a new set of holes in the array stack can be filledfrom the same microtiter plate or the syringe array can be washed anddifferent set of holes filled from a different microtiter plate.

All of the filling examples given above may be performed in parallel ona single array stack with multiple capillary tubing arrays filled withliquids from different microfiter plates or the same microfiter plate.The time to transfer fluid from chemical libraries stored in 96 and 384well microtiter plates with these methods can be quite rapid. Assuming,for example, that 100 arrays with 10,000 through-holes per array are tobe filled with liquid stored in 96 well plates, and that each transferoperation (plate exchange, fluid loading and transfer) takesapproximately 20 seconds, then it would take approximately six hours tofill all of the arrays. The filling operations can take place in anenvironmental controlled chamber (temperature, humidity, atmosphere,etc.). The invention also provides a method for screening compoundlibraries to predict the ability of each compound to be absorbed by apatient.

Transfer from a Microtiter Plate with an Array of Flexible Members

As now described with reference to FIGS. 12 b and 12 c, fluid can betransferred from individual wells 112 of a microtiter plate 114 with anarray 140 of flexible members 142, e.g., shape memory alloy fibers. Thefiber diameter is equal to or less than the inside dimension of thethrough-holes 12 in the array 10 into which fluid will be transferred.The number of fibers in the bundle may, for example, be equal to thenumber of wells in the microtiter plate 114. The ends of the fibers atone end of the bundle can have a center-to-center spacing equal to thespacing of the holes in the through-hole array, while the ends of thefibers at the opposite end can have a center-to-center spacing equal tothe spacing of wells in the microtiter plate. The fibers can be held inplace with a series of through-hole jigs designed to increase thespacing between fibers from one end of the bundle to another. Once fixedin place, shape memory alloy fibers can be heated above their criticaltransition temperature to make the imposed fiber curvature permanent.After they are cooled to room temperature, the fibers can be removedfrom the holding jig, with the change in fiber center-to-center spacingintact. The close packed end of the fiber bundle can then be insertedinto the through-hole array into which fluid from each well in the plateis to be placed. The opposite end can be arranged such that each fiberis positioned above a well in the microtiter plate, and the ends of thefibers can be immersed in the fluid contained in each well. Onretraction of the fiber bundle from the microtiter plate, as shown inFIG. 12 c, a small volume drop 144 remains attached (e.g., by surfacetension) to the end of each fiber 142. A force may be applied to theopposite end of the fiber bundle to pull the bundle through the holes 12of the through-hole array 10, such that the fluid is brought intocontact with the corresponding through-holes. As the fibers 142 arepulled through the hole 12, surface tension acts to hold the liquid inthe through-hole as the fiber is removed.

Successive Dilution

Addressable loading may also be employed to fill a series ofthrough-holes with different and specified concentrations of the samesolute. A chosen series of through-holes is filled with a quantity ofsolvent, denoted Z nanoliters (nL), either by dipping the holes into thesolvent or by dispensing the solvent from a microsyringe 232 (shown inFIG. 21), or other fluid transfer device. This is shown as the firststep 120 in the flow chart of FIG. 13. In step 121, microsyringe 232, oranother fluid transfer device, is filled with an X molar solution of thesolute, and is then positioned over the first through-hole. Y nL of thissolution, where (Y+Z) nL is sufficient volume to overfill the hole andto create positive menisci, is expelled such that it forms a droplet atthe end of the syringe tip. The syringe tip is lowered until the solventdroplet contacts the surface of the solution, causing the two liquids tomix 122 and produce a solution of concentration YX/(Y+Z) molar. Theouter surface of the syringe tip and the faces of the array must benonwetting toward the solution being dispensed. The syringe plunger isthen withdrawn in step 123 to suck up Y nL of the diluted solution. Thesyringe tip is positioned above the next (N+1) through-hole 124 and Y nLof the diluted solution is dispensed into the solvent to dilute 125 byanother factor of Y/(Y+Z). The process is repeated so as to dispensesolution into a series of individual through-holes, each time dilutingby Y/(Y+Z).

Chemical Gradient Methods

In accordance with preferred embodiments of the invention, a particularchemical species is not loaded uniformly into all the holes of an array,but, rather, a gradient of chemical species is created in at least onedimension in a two-dimensional through-hole array. As used in thisdescription and in any of the appended claims, the term “gradient”refers to its ordinary mathematical meaning, i.e., a variation, alongone or more directions, of a specified quantity. The directions, here,are taken along the surface of an array of through-holes. Thus, agradient in the concentration of a specified chemical species in thethrough-holes may be said to exist with respect to a particulardirection, or, for that matter, with respect to various directions.Thus, a particular gradient may be, but need not be, monotonic, and theconcentration of a species, in a specified direction, may rise and fall.

Referring now to FIGS. 9 a-9 c and 14 a-14 c, dip loading methodsdescribed above may be used for creating a concentration gradient in anarray of through-holes. In accordance with such methods, the array isdipped into a chemical solution at a controlled rate such that thechemicals in the solution have different amounts of time to mix with, orreact with, substances by diffusion into various through-holes.

After an array has been filled, by dip loading, with first liquid 82, asdiscussed above with reference to FIGS. 9 a-9 c, plate 10 issubsequently lowered into a second container 84 containing a secondliquid 86, as shown in FIG. 14 a. Since the through-holes of plate 10are now filled with the first liquid 84, the second liquid 86 maydiffuse into, mix with, displace, or otherwise react with, the contentsof through-holes 12. Each of these processes is characterized by a rate.Instead of fully immersing the plate until the particular reaction,physical or chemical, runs to completion, plate 10 may be partiallyimmersed into second liquid 86, as shown in FIG. 14 b, for a specifiedduration of time, and then withdrawn, wholly or partially, as shown inFIG. 14 c. If plate 10 is reimmersed in liquid 86, to a different depth88 and for a newly specified duration of time, certain of thethrough-holes 12 will contain material subject to differential physicalor chemical processes. Thus, a gradient will have been created withrespect to the direction z along the array of through-holes. Naturally,this process may be repeated or otherwise modified to create a specifiedgradient.

In accordance with alternate embodiments of the invention, other meansmay be employed in order to spatially modulate the concentration of aspecified species allowed to diffuse into a through-hole located at aparticular position within the array. Referring to FIG. 14 d, a sideview is shown of through-hole array plate 10 immersed in liquid 86.Diffusion of liquid 86 into through-holes 12 is modulated by membrane orfilter 87, for example, that, by virtue of its tapered shape orotherwise, gives rise to a gradient of a specified species with respectto the position of a through-hole within the array.

In a related example, by slowly lowering a through-hole array filledwith a gel forming solution thin-edge first into a polymerizationinitiating mixture different gel densities may be obtained within inholes. By rotating plate 10 by 90° about axis y transverse to thesurface of the plate, and then slowly dipping into a reagent thatderivatizes the gel with a free cationic moiety, and then rotatinganother 180° about y and slowly dipping into a reagent that derivatizesthe gel with an anionic moiety, a two-dimensional size/charge selectionmatrix is created that is useful for separating protein mixtures in amanner similar to a 2-D polyacrylamide gel, but with the advantage ofgreater separation speed and greater access to the separated proteins.

In another embodiment, concentration gradients within through-holearrays are use to optimize the concentrations of A and B in atwo-component reaction between Reagent A and Reagent B. The mixingprocess is described in greater detail below. Referring to FIG. 15, afirst through-hole array, shown in cross section and designatedgenerally by numeral 90, is loaded with Reagent A using a chemicalgradient method that results in the through-holes along one row, in thex direction, of the array will have the same concentration of A and theconcentration gradient is along the array columns, in the z direction. Asecond array, designated generally by numeral 92, is loaded with ReagentB such that the concentration gradient is along the rows (x direction)of the array and through-holes along a column (z direction) of the arrayhave all the same concentration. Bringing the two arrays in contactcauses mixing between aligned through-holes 12. Along each row or columnof the combined arrays the concentration of A relative to B in themixture changes in a regular and known manner. Along the array diagonal,the concentrations of A and B in the mixed liquids are equal and butchanging in overall concentration.

Variants from the basic scheme for creating a known two-dimensionalgradient in the relative proportions of A and B include loading the Aand B arrays by dipping along a diagonal, by the orthogonal combinationof two arrays each with a 2-D gradient of A relative to B or by dilutionwith arrays loaded with Reagent A or B where some of the through-holesare empty. Analysis of the through-hole contents readily determinesoptimal reaction conditions since the reaction conditions in eachthrough-hole are known. Gradients created in the solution with respectto quantities other than solute concentration are also to be understoodto be within the scope of the present invention, and may include,without limitation, such characteristics as temperature, electric field,magnetic field, etc.

Mixing and Dilution

As discussed in greater detail in the following sections, microwellplates may advantageously be stacked for such purposes as mixing ordilution. One such application is the apportionment among through-holesof a sample of cells. Considering, for purposes of example only,through-holes on a single plate that are 250 μm square and 500 μm deep.When three such microchannel plates are stacked (as would be the case ina 2-step assay), the total volume of a single channel (i.e., thecombined volume of three through-holes) is ˜100 nL. If the entirechannel is filled from a dense yeast cell culture (˜10⁷/mL) each channelthen contains approximately 10³ yeast cells. Based upon a yeast cellvolume of 70 μm³,the maximum number of cells per 100 nL channel is onthe order of 10⁶, consonant with a typical minimum of 100 cells permicrochannel is required to compensate for variable yeast cell responseto the bioassays.

Referring to FIGS. 15-19, mixing and dilution are shown between thecontents of two substantially planar through-holes array plates 90, 92(i.e., array plates having neither flanges nor indentations). In FIG.15, in particular, a cross-sectional view is shown of portions of twothrough-hole arrays 90, 92. Both the top and bottom surfaces of eachplaten are hydrophobic, as discussed in detail above. The through-holewalls 24 are preferably hydrophilic. Through-holes 12 of both platensare overfilled with high surface energy fluids 150, such as aqueoussolutions, for example, such that each through-hole 12 has apositively-curved meniscus 152 protruding above the platen surface 154.

Alternatively, the platen surfaces 154 may be hydrophilic and asufficient amount of a low surface energy fluid, such as an alkane, forexample, is loaded into the through-holes 12 to form positive menisci152.

FIG. 16 shows a cross-sectional view of the two platens 90, 92 after thesurfaces of the liquids 150 in co-registered through-holes 12 arebrought into contact. The release of the surface tension drivesconvective mixing between fluids contained in the opposite platens 90,92. Once the two surfaces of the platens themselves have been broughtinto contact, each set of two-coregistered through-holes forms a longerchannel 170, as shown in FIG. 17. Mixing proceeds within the longerchannels by a combination of convection and statistical diffusion.

Referring to FIG. 18, in cases where through-holes 12 of one or both ofarrays 90, 92 are underfilled (such that there is an air-gap separatingthe fluid 180, 182 when the plates are contacted), the application ofslight positive pressure, designated by arrows 184, may be employed tobring the surfaces of the two fluids into contact while a small gap 188is still present between the surfaces of the two platens 90, 92.

In yet other embodiments of the invention, with reference to FIG. 19,first platen 90 is filled with sufficient fluid 150 to form positivemenisci 190, 192, while a second platen 92 is filled with an amount offluid 194 that is insufficient to form positive menisci, but sufficientto make contact with the surface 190 of the menisci of the first platenwhen the two platens are stacked. Mixing proceeds by bringing the liquidsurfaces into contact as previously described.

Control of Environmental Factors

In accordance with embodiments of the invention, methods and apparatusare provided for maintaining high relative humidity levels (typicallyabove 95%) in the environment surrounding through-hole arrays duringvarious operations of the invention. Maintenance of high humidity levelsmay advantageously minimize, if not eliminate, evaporation of solutionscontained in the through-hole arrays. The level of humidity required tokeep the volume loss at an acceptable level depends upon the length oftime needed to perform the various desired operations, as well as onsuch factors as the ambient temperature, and the volume of fluidcontained in each through-hole. At a temperature of 21° C., theevaporative loss expected from a droplet of water may be predicted inaccordance with Fick's law, as depicted in FIG. 20. The time, inseconds, for 10% evaporative loss is plotted for a 50 μL droplet (bycurve 200) and for a 50 nL droplet (curve 202) as a function of relativehumidity. Unless humidity levels are close to 100%, the smaller dropletwill evaporate very rapidly, with a 10% mass loss in 11 seconds at 65%relative humidity.

Humidity Control during Addressable Loading and Unloading

With reference to FIG. 21, and apparatus is provided for advantageouslyreducing the volume of the enclosed environment subject to humiditycontrol. In accordance with embodiments of the invention, a through-holearray 10 is mounted above a reservoir 210 of water. A 96-well, 384-wellor higher density microtiter plate 212 is mounted above the samereservoir proximal to the through-hole array. Reservoir 210 may containa water absorbent material such as a sponge 214 to keep the water fromsloshing when the reservoir is moved. An optically transparent plate 216is placed on top of the reservoir separated from the walls of thereservoir by a thin layer 218 of a viscous non-hydroscopic fluid such assilicone grease, for example. One or more small holes 220 are drilledthrough transparent plate 216. Through each hole a microsyringe needle222, microcapillary, pin, cannula, or other fluid transfer element isinserted.

Computer control of motorized translation stages may be provided, suchas along axis y to move the fluid transfer element 224 towards and awayfrom the top face 226 of the through-hole array 10. Fluid transferelement 224 may include syringe 232 with plunger 234 fluid transferelement 224, for example. Additionally, the arrays and reservoir may bemoved in the plane of the x and y axes, preferably by motorized stage228 under control of controller 230, with respect to the fluid transferelement. If indicated, further computer-controlled stages may beprovided, for example, to actuate the fluid transfer device. Transfer ofmaterials between the microtiter plate and through-hole array proceed bymeans described above in reference to FIGS. 10-12.

Additional reservoir(s) containing solution(s) for cleaning/sterilizingthe fluid transfer element may also be provided within the confines ofchamber 236 which encloses the apparatus heretofore described.Alternatively the water in the large reservoir 210 may be used for thispurpose. An illumination source 238 may also be provided for toilluminate the array for optical inspection during loading/unloading,either visually or by means of a video camera 240.

Humidity Control During Dip Loading and Mixing

An environment enclosure 236, as shown in FIG. 21, may also be utilizedin order to preventing evaporation from the through-hole arrays duringdip loading and mixing. Sealable chamber 236 is large enough to containthe array and necessary apparatus for performing such desired operationsas are required, including, for example, motors and translation stages228, as well as an alignment jig 156 (shown in FIG. 15) for mixingoperations.

Cool water vapor is generated by an external humidifier and injectedinto the chamber through a port. An ultrasonic humidifier is able togenerate enough vapor to maintain humidity levels above 95% in a 0.13cubic meter chamber. A circulation system consisting of a fan andbaffles is provided to distribute the water vapor uniformly throughoutthe chamber. Various doors, hatches and iris ports may be provided foraccessing the interior of the chamber. The temperature within the boxmay be controlled by a resistive heater and an electronic temperaturecontroller. Such a chamber may be assembled by attaching a secondhumidifier to a commercial infant incubator.

Any surface which must be kept dry, including optical windows, andcorrodible metals may be heated slightly above the ambient to preventcondensation. A humidity sensor is also provided to monitor humiditylevels inside the chamber. All computer and electronic hardware areplaced outside the chamber and are connected to components inside thechamber via a wire feedthrough.

The through-hole array may be filled within the chamber, or loadedexternally and transferred into to the chamber in a humidified sealedcassette.

Humidity Control during Optical Analysis and Transfer between LoadingStations

Referring now to FIG. 22 a, an exploded perspective view is provided ofa compact portable cassette, designated generally by numeral 250, formaintaining relative humidity levels above 95% when the array isremoved, for optical analysis, for example, from the humidified loadingchamber 236 (shown in FIG. 21). FIG. 22 b provides a side view, in crosssection, of the humidified cassette 250 of FIG. 22 a. Through-hole array10 and an aqueous microlens array 252 are mounted in a compact, sealableenclosure. The though-hole array is positioned and held in place bymeans of alignment pins 256 or some other means. Both the array mount258 and cover holder 260 are covered by an optically transparent plate262 secured to the mount and cover by adhesive or other mechanicalmeans. The mount may also include magnets, pins, grooves or otherphysical features to facilitate position the array inside opticalanalysis equipment.

The humidity is raised by passive evaporation from water contained in atransparent glass microcapillary bundle 252 (or, ‘microchannel array’)mounted beneath the through-hole array and secured by set screws or someother mechanical means. (Glass microcapillary bundles are manufacturedby Schott Fiber Optics, Southbridge, Mass.). Typical dimensions for eachcapillary 264 are diameter of 200 microns and depth of 1 mm. The top 266and bottom 268 of the microchannel array 252 are made hydrophobicaccording to the procedures described above. The arrangement of thecapillaries 264 in the array need not be regular. Water 270 in thecapillaries forms a set of microlenses which serve to diffuse light fromlight source 272 across the array and thus provide a uniformillumination field for optical analysis. The use of liquid microlensarrays, generally, is within the scope of the present invention.

Another advantage of the aqueous lens array is that the water is held inplace by surface tension. Thus the operator need not keep the humiditycassette level, or avoid applying the typical accelerations that occurwhen the plate is carried around a room and placed on horizontalsurfaces. Also the cassette may be agitated during incubation to promotecell growth inside the arrays.

Control of Other Environmental Factors

Pressure, light and temperature, are controlled by enclosing the arrayin an appropriately constructed chamber and then controlling theenvironment inside the chamber by conventional means. To preventevaporation such chamber must also be equipped to maintain high relativehumidity.

Having thus described various illustrative embodiments of the presentinvention and some of its advantages and optional features, it will beapparent that such embodiments are presented by way of example only andare not by way of limitation. Those skilled in the art could readilydevise alternations and improvements on these embodiments, as well asadditional embodiments, without departing from the spirit and scope ofthe invention. All such modifications are within the scope of theinvention as claimed.

1. A method for loading a plurality of disparate sample containers, thesample containers comprising an integral structure, such that theconcentration of a specified substance in each container ischaracterized by a gradient with respect to position of the containerwithin the structure, the method comprising: a. introducing a firstliquid into the disparate containers; and b. contacting the containerswith a second liquid, the second liquid containing the specifiedsubstance, in such a manner that the degree of diffusion of thespecified substance into the disparate containers differs in acontrolled manner among the containers, wherein contacting thecontainers with the second liquid includes differing a duration ofcontact with the second liquid as among the disparate containers.
 2. Amethod for loading a plurality of disparate sample containers, thesample containers comprising an integral structure, such that theconcentration of a specified substance in each container ischaracterized by a gradient with respect to position of the containerwithin the structure, the method comprising: a. introducing a firstliquid into the disparate containers; and b. contacting the containerswith a second liquid, the second liquid containing the specifiedsubstance, in such a manner that the degree of diffusion of thespecified substance into the disparate containers differs in acontrolled manner among the containers, wherein contacting thecontainers with the second liquid includes modulating the diffusion rateof the specified substance into the containers.
 3. A method according toclaim 2, further comprising the step of creating a gradient of thespecified substance in the second liquid prior to contacting thecontainers with the second liquid.
 4. A method according to claim 3,wherein the step of creating a gradient of the specified substanceincludes applying electrophoresis.
 5. A method according to claim 2,wherein the step of modulating the diffusion rate includes contactingthe containers with the second liquid through a membrane having apermeability variable with respect to position.
 6. A method for loadinga liquid into a plurality of through-hole platens, each platen having aplurality of through-holes, the method comprising: a. stacking at leasttwo substantially empty platens together in such an adjacent manner thatat least one of the plurality of through-holes from each platen isregistered with a through-hole of each other adjacent platen so as toform a registration set of through-holes unimpeded by any solidstructure; and b. transferring the liquid to form a continuous column ofliquid in each registration set.
 7. A method according to claim 6,wherein each platen is separated from each adjacent platen by an airgap.
 8. A method according to claim 6, wherein the step of transferringliquid includes transporting the liquid through capillary tubes.
 9. Amethod according to claim 6, wherein the step of transferring liquidincludes transporting the liquid through at least one cannula.
 10. Aperforated platen having substantially parallel planar surfaces formanipulating distinct liquid samples, each sample having a volume lessthan 1 microliter, the platen comprising: a. an inner layer ofhydrophilic material; b. two outer layers of hydrophobic materialcoupled to opposite sides of the inner layer; c. a two-dimensional arrayof through-holes including, at least a first hole having a first liquid,and d. at least one hole, adjacent to the first hole, containing asecond liquid distinct from the first liquid, the through-holes eachhaving a diameter less than 400 micrometers and a density of at least1.6 through-holes per square millimeter, wherein the two outer layers ofhydrophobic material prevent outmigration of the first and secondliquids.